The ribosomal protein S7, a primary 16S rRNA-binding protein, plays an essential role in stabilizing the 3' major domain of 16S rRNA and also in feedback regulation of the str operon, as a translational repressor. We examined amino acid residues in ribosomal protein S7 from Bacillus stearothermophilus (BstS7) that are essential for 16S rRNA binding. Truncation of the N-terminal 10 residues of BstS7 abolished its binding to 16S rRNA, whereas removal of the C-terminal eight residues had no effect on the binding activity. Subsequently, we used site-directed mutagenesis to identify essential basic residues in the N-terminal region for 16S rRNA binding. Mutation of Agr3 and Lys8 significantly weakened the binding activity, and a smaller decrease in binding activity was observed with Arg2 and Arg9 mutations. These observatiOnS indicate that N-terminal basic residues, especially Arg3 and Lys8, play a crucial role as positively charged recognition groups for the negatively charged phosphate backbone of 16S rRNA. In addition, the mutagenesis study showed that Arg75, Arg75, Arg94, and Arg101 which are located in a concavity formed by the β-ribbon arm and the α-helix (α4) individually make only a small contribution to 16S rRNA binding, but together probably form a positively charged binding site for 16S rRNA. With regard to aromatic residues, Tyr84 on the tip of the β-ribbon arm was found to be involved in 16s rRNA binding, whereas the conserved aromatic residues Trp102 and Tyr106 in the concavity had little effect. We then probed the 16S rRNA-binding site(s) for the N-terminal region of S7 with iron revered to the mutant of BStS7 containing a single cysteine residue at position 4. The N-terminal region of S7 is placed in close proximity to helix 43 in the 16S rRNA. Probing also revealed also regional cleavages between nucleotides 1397 and 1438, near the P-site region in 16S rRNA. This finding is consistent with a three-dimensional model of 16S rRNA that shows close proximity of helix 43 to the P-site during three-dimensional folding.
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